Caspase-8 induces cleavage of gasdermin D to elicitpyroptosis during Yersinia infectionJoseph Sarhana,b,c,1, Beiyun C. Liua,c,1, Hayley I. Muendleinc,d, Peng Lie, Rachael Nilsonc, Amy Y. Tangc,Anthony Rongvauxf, Stephen C. Bunnellc, Feng Shaoe, Douglas R. Greeng, and Alexander Poltorakc,h,2

aGraduate Program in Immunology, Tufts University Sackler School of Biomedical Sciences, Boston, MA 02111; bMedical Scientist Training Program, TuftsUniversity School of Medicine, Boston, MA 02111; cDepartment of Immunology, Tufts University School of Medicine, Boston, MA 02111; dGraduate Programin Genetics, Tufts University Sackler School of Biomedical Sciences, Boston, MA 02111; eCollaborative Innovation Center for Cancer Medicine, NationalInstitute of Biological Sciences, 102206 Beijing, China; fProgram in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle,WA 98109; gDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38104; and hCenter of High Biomedical Technologies,Petrozavodsk State University, Petrozavodsk, Republic of Karelia, 185910, Russia

Edited by Eicke Latz, University of Bonn, Bonn, Germany, and accepted by Editorial Board Member Peter Palese October 7, 2018 (received for review June5, 2018)

Cell death and inflammation are intimately linked during Yersiniainfection. Pathogenic Yersinia inhibits the MAP kinase TGFβ-activated kinase 1 (TAK1) via the effector YopJ, thereby silencingcytokine expression while activating caspase-8–mediated celldeath. Here, using Yersinia pseudotuberculosis in corroborationwith costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol,a small-molecule inhibitor of TAK1, we show that caspase-8activation during TAK1 inhibition results in cleavage of both gas-dermin D (GSDMD) and gasdermin E (GSDME) in murine macro-phages, resulting in pyroptosis. Loss of GsdmD delays membranerupture, reverting the cell-death morphology to apoptosis. Wefound that the Yersinia-driven IL-1 response arises from asyn-chrony of macrophage death during bulk infections in which twocellular populations are required to provide signal 1 and signal 2 forIL-1α/β release. Furthermore, we found that human macrophagesare resistant to YopJ-mediated pyroptosis, with dampened IL-1βproduction. Our results uncover a form of caspase-8–mediatedpyroptosis and suggest a hypothesis for the increased sensitivityof humans to Yersinia infection compared with the rodent reservoir.

pyroptosis | caspase-8 | gasdermin | Yersinia | TAK1

Cell death and inflammation are critical regulators of immuneresponses which must be carefully balanced to ensure host

survival during infection. Upon initial pathogen contact withinnate immune cells, the host cell signaling through Toll-like re-ceptors (TLRs) simultaneously engages a cell’s intrinsic prosurvivalprogram and produces inflammatory cytokines to propagate thealarm of infection (1). These cytokines are critical for the activationand recruitment of further waves of innate and adaptive immunity aswell as for reinforcing the inflammation loop via autocrine signalingin the current population. Successful pathogens have thereforeevolved ways to evade host cytokine induction to survive and repli-cate silently among host immune cells. This is done either by mod-ifying surface pathogen-associated molecular patterns that can-not be recognized by macrophage TLRs or via active blockade ofmacrophage-signaling pathways downstream of TLR activation (2).To counteract the latter, these same prosurvival, procytokine

signaling pathways also have built-in switches to engage celldeath when the cell perceives a block to its ability to producecytokines (3). One of the best-characterized examples of such aswitch lies downstream of TNF receptor. TNF receptor signalingengages the TNF receptor-associated adaptor TRADD to recruitRIP1 via death domain (DD) interactions (4), forming complex I.Complex I formation results in the activation of the MAP kinasesand NFκB, which drive the production of inflammatory cytokinesand prosurvival factors (5). When MAP kinases and/or NFκBsignaling are blocked, RIP1 dissociates from TNF receptor tofacilitate the formation of complex IIa by interacting with FADDand pro–caspase-8. Complex IIa leads to caspase-8 activation andcell death (6). If caspase activity is blocked, the signaling complex

transitions to complex IIb, in which RIP1 engages RIP3 to activatean MLKL-dependent necrotic cell-death mechanism known as“necroptosis” (7). In recent years necroptosis has similarly beenshown to occur downstream of TLR signaling (8).The success of MAP kinase and NFκB signaling thus are critical

determinants of the life and death of a cell. MAP kinase andNFκB pathways diverge from a single protein complex consistingof TGFβ-associated kinase (TAK1), TAB1, and TAB2 in whichTAK1 experiences constitutive phosphorylation and baseline ac-tivity (9). In mice, whole-body TAK1 deficiency results in lethalitybetween E9.5–E10.5 (10). Follow-up studies elucidated that TNFstimulation in the context of TAK1 deficiency leads to cell deathvia caspases (complex IIa) or, in the case of caspase inhibition, tocell death by necroptosis (complex IIb) (11, 12). Similarly, whole-body RIP1 deficiency results in perinatal lethality in which Rip1−/−

and conditionally null animals show exquisite sensitivity to tonicTNF-induced cell death (5, 13).Given the intertwining relationship between cytokine pro-

duction and cell death during infection, the central question thusbecomes what is achieved upon cell death if cytokines, the uni-versal alarms, cannot be adequately made in the first place. Thewell-studied bacteria of the Yersinia species may lend an answer.

Significance

Here we demonstrate that Yersinia YopJ-induced murine mac-rophage death involves caspase-8–induced cleavage of bothgasdermin D (GSDMD) and gasdermin E (GSDME). The ensuingcell death is rapid, morphologically is similar to pyroptosis, andinduces IL-1 release. Recently, both GSDMD and GSDME werereported to be critical effectors of caspase-1/11–driven pyroptosisand caspase-3–dependent secondary necrosis, which promptedthe redefinition of pyroptosis as cell death-mediated by gasderminactivation. Our work extends these studies and shows that acti-vation of caspase-8 in the context of TAK1 inhibition results incleavage of both GSDMD and GSDME, leading to pyroptotic-likecell death. Further study will be needed to determine whethercaspase-8 cleaves GSDMD directly or via intermediate substrates.

Author contributions: J.S. and B.C.L. designed research; J.S., B.C.L., H.I.M., P.L., R.N., andA.Y.T. performed research; A.R. and F.S. contributed new reagents/analytic tools; J.S.,B.C.L., S.C.B., and D.R.G. analyzed data; and J.S., B.C.L., and A.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. E.L. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1J.S. and B.C.L. contributed equally to this work.2To whom correspondence should be addressed. Email: Alexander.Poltorak@tufts.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809548115/-/DCSupplemental.

Published online October 31, 2018.

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Yersinia species express a number of Yersinia outer proteins(Yops) that manipulate various macrophage cytosolic processes(14). One such protein, YopJ, inhibits TAK1 via acetylation (15)with consequent induction of macrophage cell death (16). Yersinia-induced cell death was recently shown to be caspase-8 dependentbut RIP3 independent. Paradoxically, while Rip3−/−Casp8−/− mac-rophages are protected from cell death, the animals succumb to theinfection, with higher bacterial burden in various solid organs (17).Similarly, Yersinia-induced macrophage cell death is strongly de-pendent on RIP1 kinase activity; RIP1 kinase-inactive animals alsoshow increased sensitivity to infection (18). These studies suggestthat macrophage death in the context of Yersinia infection is pro-tective for the animal host.Interestingly, despite the ability of YopJ to dampen cytokine

production, Yersinia infections in mice elicit a robust IL-1 re-sponse, with IL-1 receptor signaling playing a crucial role in thesurvival of the animal (19). IL-1 maturation requires the formationof a caspase nucleation event termed the “inflammasome.” Theinflammasome is triggered via the sensing of a cytosolic “danger”signal that nucleates multiple units of procaspase-1 for proximity-induced autocleavage and activation. One of the best-studiedinflammasomes is the NLRP3 inflammasome, which nucleates inresponse to bacterial components, crystals, or potassium efflux.NLRP3 recruits apoptosis-associated speck-like protein (ASC),which then recruits procaspase-1 via homotypic CARD-domainbinding (20). Caspase-1 is activated via autocleavage by which ac-tive caspase-1 cleaves pro–IL-1β and pro–IL-18. The release ofmature IL-1β in conjunction with inflammasome-driven celldeath is termed “pyroptosis” (11). With the discovery that caspase-11 drives pyroptosis via activation of the pore-forming proteingasdermin D (GSDMD) (12), the NLRP3 inflammasome wasfound to activate as a consequence of plasma membrane ruptureand the concurrent efflux of potassium ions (21). More recently,activation of the NLRP3 inflammasome has also been ob-served during necroptosis (22), suggesting that inflammasomeformation may be a commonality shared among many necrotic formsof cell death.In this work, we set out to investigate the morphological features of

the caspase-8–driven cell death during TAK1 inhibition, using Yersiniapseudotuberculosis as our infection model. To model a complex in-fection system to minimal manipulable units, we additionally useLPS and 5Z-7-Oxozeaenol (5z7), a potent small-molecule inhibitorof TAK1 (23), to mimic the basic cell-death phenotype inducedby Yersinia. We found that Yersinia-induced cell death bears astriking morphologic resemblance to pyroptosis, the involvementof multiple gasdermins, and the potency of IL-1 production. Thisled us to conclude that caspase-8 can drive pyroptosis in cases ofTAK1 inhibition during inflammatory signaling.

ResultsDuring TAK1 Inhibition, TLR Stimulation Drives Caspase-8–DependentCell Death with Necroptosis as a Backup Mechanism. Yersinia species,including Yersinia pestis and Y. pseudotuberculosis, induce macro-phage cell death via effector YopJ inhibition of the level-threeMAP kinase TAK1 (15). We used nuclear incorporation of propi-dium iodide (PI) to monitor the kinetics of cell membrane integrityloss in real time. We see that in B6 macrophages YopJ-specificmacrophage death occurs is complete by 4–6 h post bacterialchallenge and that macrophages lacking both RIP3 and caspase-8are protected (SI Appendix, Fig. S1.1A), consistent with publishedreports (17). To investigate the individual contribution of caspase-8 and RIP3, we used the small-molecule inhibitors zVAD for pan-caspase inhibition or Nec1 for inhibition of necroptotic machinery.We find that Y. pseudotuberculosis-driven cell death can be partiallyrescued by inhibition of caspases or RIP kinases; however, completeablation of Yersinia-driven cell death requires the addition of bothzVAD and Nec1 (Fig. 1A, Left). We can effectively mimic this re-sponse by costimulation of macrophages with the small-molecule

inhibitor of TAK1 5z7 in combination with a TLR agonist, in thiscase LPS (Fig. 1A, Right and SI Appendix, Fig. S1.1B).Necrostatin-1 (Nec1), the small-molecule inhibitor of

RIP1 kinase activity, is used to block necroptosis driven by RIP1/RIP3 and the effector of necroptotic cell death, MLKL (24).Given the partial protection with Nec1, we investigated thecontribution of necroptotic players during Yersinia-driven as wellas LPS/5z7-driven cell death. Using genetic knockouts of RIP3and MLKL, we found that Mlkl−/− and Rip3−/− macrophages arefully susceptible to Yersinia- and LPS/5z7-driven cell death (Fig.1B and SI Appendix, Fig. S1.1C). With the addition of zVAD,Rip3−/− and Mlkl−/− macrophages become protected from death,indicating that in the context of TAK1 inhibition necroptosis isengaged only when caspase activity is blocked. However, the fullsusceptibility of RIP3- and MLKL-deficient cells to Yersinia-driven death argues that necroptosis is not the primary path-way of cell death during Yersinia infection. The addition ofNec1 during Yersinia challenge or LPS/5z7 stimulation showspartial protection regardless of the presence of RIP3 or MLKL.Working upstream in the pathway, RIP1 kinase activity plays

critical roles for the initiation of necroptosis during caspase inhibition(25). However, RIP1 also has a role in TNF-driven apoptosis via theformation of complex II (6). We found that RIP1 kinase-inactive(RIP1K45A/K45A) macrophages are partially protected from Yersinia-and LPS/5z7-driven cell death, recapitulating the effect of Nec1 ad-dition (Fig. 1C) (18). RIP3 kinase-inactive (RIP3K51A/K51A) macro-phages behave like Rip3−/− macrophages (SI Appendix, Fig. S1.1D),and RIP1/3 double-kinase–inactive macrophages show susceptibilitysimilar to that of RIP1 single-kinase macrophages (Fig. 1C and SIAppendix, Fig. S1.1E). RIP1 thus plays an enhancement role indriving cell death during Yersinia infection but is not the criticalfactor. Confirming previous findings (17), cells deficient for bothRIP3 and caspase-8 are fully protected from Yersinia- and LPS/5z7-driven cell death (Fig. 1D). We expand on this finding and show thatmacrophages lacking RIP3 and FADD are equally resistant toYersinia-induced and LPS/5z7-induced cell death (Fig. 1E). Giventhat Rip3−/− macrophages are fully sensitive to cell death, the fullprotection provided by the additional loss of caspase-8 or FADDindicates that caspase-8/FADD complex is necessary to drive celldeath. Yersinia as well as TLR stimulation under TAK1 inhibitionthus drives a mode of rapid, cell death that is caspase-8 driven, with apartial contribution from RIP kinase 1.TAK1 kinase activity is critical for the activation of the MAP

kinase- and NFκB-signaling pathways. To address the individualroles of MAP kinases and NFκB in TAK1-induced cell death, westimulated B6 macrophages with LPS in the presence of variousMAPK and IKKβ inhibitors. Treatment of LPS-stimulated cellswith p38, JNK, or MEK1/2 inhibitors individually was not suffi-cient to induce cell death to the extent observed in cells stimulatedwith LPS + 5z7 (SI Appendix, Fig. S1.2A). However, treatment ofLPS-stimulated cells with a combination of p38 and JNK inhibi-tors resulted in cell death more potently than induced by eitherinhibitor independently (SI Appendix, Fig. S1.2A). Treatment ofLPS-stimulated macrophages with various IKKβ inhibitors inducescell death in a manner similar to TAK1 inhibition (SI Appendix, Fig.S1.2B). Both MAPK inhibition- and NFκB inhibition-dependentcell death are similarly absent in RIP3−/−Casp8−/− macrophages(SI Appendix, Fig. S1.2 A and B).Using TLR adaptor-deficient bone marrow-derived macro-

phages (BMDMs), we found that LPS + 5z7-induced cell deathcan occur independently of the TLR4 adaptor MyD88 and isfully sufficient in the setting of adequate TRIF signaling (SIAppendix, Fig. S1.3A). Interestingly, in the case of TRIF, LPS +5z7 deficiency still induced cell death via MyD88 signaling, butthe character of this cell death is different. The percentage of celldeath is lower in TRIF deficiency, and, interestingly, the celldeath is entirely dependent on RIPK activity, given its re-versibility with Nec-1. To explore further the role of other TLR

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adaptors, we stimulated B6 BMDMs stimulated with variousTLR agonists or an SMAC mimetic in combination with 5z7 (SIAppendix, Fig. S1.3B) and observed faster kinetics of cell deathwith TLR stimulation or SMAC mimetic treatment than with5z7 alone. Interestingly, LPS is unique in that it induces celldeath in a TNF-independent manner (SI Appendix, Fig. S1.3B),whereas cell death induced by other TLR agonists + 5z7, SMACmimetic + 5z7, or 5z7 alone is strongly TNF dependent. In-terestingly, these TNF-dependent forms of cell death are also

strongly dependent on RIP kinase activity, as indicated by theelimination of cell death with the addition of Nec-1. Much liketype-I IFN, TNF is produced in resting cells (26, 27). The phe-nomenon of tonic TNF production likely underlies the basis ofcell death in the setting of cell death induced by 5z7 alone.

TAK1 Inhibition Results in Externalization of Cytosolic Content withPan-Caspase Cleavage. Traditionally, caspase-8 activation hasbeen associated with apoptosis, characterized morphologically by

A

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Fig. 1. During TAK1 inhibition, TLR stimulation drives caspase-8–dependent cell death, with necroptosis as a backup mechanism. BMDMs from C57BL/6 (B6)and various genetically modified animals were stimulated with Y. pseudotuberculosis IP2666 (Left) or LPS (10 ng/mL)/5z7 (125 nM) (Right). Y. pseudotu-berculosiswas infected at an MOI of 30. Percentage cytotoxicity was calculated by 4×microscopy via counting PI+ nuclei per field of view, normalized to 100%lysis by 0.1% Triton X-100. Caspases were inhibited with zVAD treatment (50 μM), and RIP kinase activity was inhibited with Nec1 (10 μM). All inhibitors wereadded simultaneously at time 0. The cytotoxicity curve of each genotype is superimposed over B6 cytotoxicity curves (gray) for clarity of comparison. Data inA–D are from one experiment utilizing the same set of B6 controls; data in E are from an independent experiment. All kinetic cytotoxicity data are repre-sentative of at least three independent experiments. See related SI Appendix, Fig. S1.

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the shedding of apoptotic bodies and the retention of membraneintegrity during cell death to promote efferocytosis over sec-ondary necrosis and the release of cytosolic contents (28).However, the rapidity of PI uptake during Yersinia- and LPS/5z7-driven cell death is suggestive of necrotic cell death. Indeed,LPS/5z7 induces rapid and indiscriminate release of cytosoliccontents into the surrounding culture medium as compared withthe canonical apoptosis stimulant etoposide and the recentlysynthesized small-molecule raptinal, which drives cell death viacaspase-9 activation of caspase-3 and -7 (Fig. 2A) (29).Probing for caspase activation and their appearance in the

culture medium (precipitated supernatant), we found that themajority of fragments corresponding to active caspase-8, caspase-9, caspase-3, and caspase-7 have accumulated extracellularly by5 h of LPS/5z7 stimulation (Fig. 2B). In contrast, these cleavedcaspase fragments can be seen by 5 h in cell lysates of etoposide-treated cells, but cleaved caspase-3 and caspase-7 are not seen inthe supernatant until more than 16 h after stimulation.Additional cytosolic contents released upon LPS/5z7 stimula-

tion include cleaved poly (ADP-ribose) polymerase (PARP),cyclophilin A, and GAPDH. Congruent with the cell-death kinetics,the apoptosis machinery is blocked with addition of zVAD, whichswitches the cell fate to necroptosis, as indicated by the appearanceof phosphorylated MLKL with zVAD addition (Fig. 2C). Usingmacrophages deficient for both RIP3 and caspase-8, we con-firmed that the release of cytosolic content during LPS/5z7 stimula-tion occurs as a consequence of cell death. With Rip3−/− Casp8−/−

macrophages, the cleavage of caspase-3 and PARP is no longerpresent beyond baseline levels, and cytosolic proteins such ascyclophilin A, GAPDH, and actin remain intracellular duringLPS/5z7 stimulation (Fig. 2D).We confirmed that 5z7 can inhibit both MAPK and NFκB

signaling in the context of LPS signaling in BMDMs (SI Ap-pendix, Fig. S2A). To rule out the contribution of cell death andto study the effects of the Yersinia effect on YopJ, we infectedRIP3−/−Casp8−/− macrophages with wild-type and YopJ-deficient(ΔJ) Yersinia. As early as 30 min after infection, wild-type Yersinia in-hibits MAP kinase activation as measured by p38 and ERK (MEK1/2)phosphorylation. In contrast, the effect of YopJ on NFκB activationas measured by IKKα/β and p65 phosphorylation was attenu-ated (SI Appendix, Fig. S2B). In addition, the degradation andreplenishment of the NFκB inhibitor IκB-α had similar ki-netics in cells infected with wild-type Yersinia infection andcells infected with ΔJ Yersinia (SI Appendix, Fig. S2B). Collec-tively, these data agree with the previous reports that YopJpreferentially blocks the activation of MAPKs rather than ofNFκB (30, 31).

Yersinia- and LPS/5z7-Driven Cell Death Resembles Pyroptosis byMorphology. Apoptosis, necroptosis, and pyroptosis are mor-phologically distinct from each other. Using time-lapse micros-copy, we followed the progression of macrophages undergoingeach of these three well-defined modes of cell death to de-termine the morphology taken by cells dying from LPS/5z7 orYersinia (Fig. 3 A–E).For a rapid form of apoptosis, we use the small molecule

raptinal, which engages caspase-9 activation (32). Raptinal-treatedcells progress to membrane rupture by 2–3 h post stimulation,thus placing it on a timescale similar to LPS/5z7-induced celldeath (Fig. 3A). Cells undergoing raptinal treatment display typ-ical signs of cell shrinkage and extensive blebbing as early as30 min post stimulation. Annexin V positivity as an indication ofphosphatidylserine externalization is rampant by 40 min, and thisphase is extended for at least 1 h. Two hours post stimulation thecell membrane ruptures as indicated by nuclear PI signal, withmorphology of the dying cell marked by multiple necrotic bubblesresembling that of secondary necrosis (33).

Recently, Gong et al. (34) showed that necroptosis, although anecrotic form of cell death, can display phosphatidylserine ex-ternalization before membrane rupture. We confirm that withLPS/zVAD-induced necroptosis annexin V binding is evident by50–60 min post stimulation in the absence of cell shrinkage asseen in apoptosis. Membrane ruffling is seen during the annexinV-positive phase, and plasma membrane rupture takes the formof one or two large necrotic bubbles per cell that coincides withnuclear PI positivity. Necroptotic cells take 50 min to 1 h fromthe onset of phosphatidylserine externalization to plasma mem-brane rupture (Fig. 3B).To trigger pyroptosis, LPS was transfected into the cytosol to

activate caspase-11 (35). Unlike necroptosis and apoptosis, cellsundergoing pyroptosis do not show overt signs of impendingmembrane rupture in the hours leading up to cell death, although

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Fig. 2. TAK1 inhibition results in necrotic cell death, exhibiting pan-caspaseactivation and the externalization of cytosolic content. (A) BMDMs werestimulated with etoposide (150 μM), raptinal (10 μM), or LPS (10 ng/mL)/5z7(125 nM) for 0–6 h. Cellular lysates (Upper) and precipitated supernatant(Lower) were run on SDS/PAGE gels, and total protein was stained by LiCORtotal protein stain. The asterisk indicates serum proteins from residual FBS inthe culture medium. (B) BMDMs were stimulated as indicated for 5 h, exceptfor etoposide, which was stimulated for an additional 16 h. Cellular lysateand precipitated supernatant were run on SDS/PAGE gels and were probedfor pro- and cleaved forms of various apoptotic caspases. (C) BMDMs werestimulated with LPS/5z7 for 5 h in the presence of zVAD (50 μM) or Nec1(10 μM) to block caspase or RIP kinase activity, respectively. Cellular lysateand precipitated supernatant were run on SDS/PAGE gels and probed forcaspases, the caspase substrate PARP, cytosolic proteins cyclophilin A (CypA),Gapdh, phosphorylated (S345) MLKL (p-MLKL), and total MLKL. (D) BMDMsfrom B6 and Rip3−/− Casp8−/− animals were stimulated with LPS, LPS/5z7, or5z7 for 5 h. Cellular lysate and precipitated supernatant were run on SDS/PAGE gels and probed for caspases, the caspase substrate PARP, cytosolicproteins cyclophilin A (CypA), Gapdh, and actin. All Western blots are rep-resentative of three or more experiments.

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a weak PI signal from the nuclei can be observed. PI nuclear signalis simultaneous with annexin V positivity as the cell balloons outinto a spherical morphology (Fig. 3C).With LPS/5z7 costimulation, dying cells retain extensive lam-

melipodia up to 10–20 min before membrane rupture. At 10 minbefore loss of membrane integrity, the cell shrinks rapidly, fol-lowed by simultaneous onset of weak annexin V positivity,blebbing, and a weak PI signal seeping into the nucleus. Within10 min of annexin V binding the cell bursts with one large,spherical balloon along with full nuclear PI signal (Fig. 3D). Theidentical progression of cell death is seen during Yersinia-induced cell death (Fig. 3E).To quantify this phenotype on a population level, images were

taken at 4× magnification with 3,000–4,000 cells captured perframe. Annexin V and PI signal intensity per cell were plotted onthe y and x axes, respectively. Representative frames at 1.5 and3 h are shown for LPS/zVAD and LPS/5z7 (Fig. 3 F and G andMovies S1 and S2). On a population level, necroptotic cells showa distinct population of annexin V+PI− cells that transition intoPI positivity (Fig. 3F). In contrast, LPS/5z7-stimulated cellsprogress from dual negative to a PI+annexin Vweak state and gainannexin V positivity over time (Fig. 3G). Our data show that withTAK1 inhibition-induced cell death, the rapidity of membranerupture curtails the process of phosphatidylserine externaliza-tion, leading to a pyroptotic-like cell-death morphology.To support our morphological analysis, we performed TUNEL

staining to detect DNA breaks indicative of the late stages ofapoptosis. Within 220 min, 5% TUNEL+ nuclei were detected inBMDMs treated with raptinal to induce apoptosis but were ab-sent after treatment in necroptotic (LPS/zVAD) and pyroptotic

(cytosolic LPS and LPS + ATP) conditions as well as aftertreatment with LPS/5z7 (SI Appendix, Fig. S3A).

Caspase-8 Induces GSDMD Cleavage to Drive Pyroptosis, CurtailingApoptosis. GSDMD and gasdermin E (GSDME) are two effec-tors of pyroptosis downstream of caspase activation (12, 36). Todetermine whether GSDMD and GSDME are involved in LPS/5z7- and Yersinia-induced cell death, macrophages were stimu-lated with various cell-death stimulants, and cell lysates wereprobed for active cleavage products of GSDMD and GSDME.Weobserved that GSDMD and GSDME are both cleaved to generatethe active p30 fragment within 3 h of LPS/5z7 stimulation as wellas after Yersinia challenge (Fig. 4A). In addition to the activep30 fragment generated by caspase-1 and caspase-11 as previouslydescribed (12, 36), we also observe a higher GSDMD fragment(∼40 kDa), likely the inactive p43 recently reported downstreamof caspase-3 activity (37). Macrophages lacking GsdmD show asignificant delay in PI incorporation during challenge with eitherLPS/5z7 or Yersinia (Fig. 4 B and C). Morphologically, the cur-tailed annexin V staining in GSDMD-sufficient cells became ro-bust and prolonged with the loss of GSDMD, accompanied by anincrease—albeit incremental—in TUNEL+ nuclei, recapitulatingan apoptotic morphology (Fig. 4 D and E and SI Appendix, Fig.S4A). This can be seen on a population view with LPS/5z7 thatdrives a synchronized cell-death response (Fig. 4D) as well as onan individual cellular basis during Yersinia infection (Fig. 4E). Lossof GSDME either singly or in conjunction with GSDMD loss didnot confer further protection against LPS/5z7-induced death (SIAppendix, Fig. S4 B and C).

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Fig. 3. Yersinia- and LPS/5z7-driven cell death resembles pyroptosis by morphology. (A–E) B6 BMDMs were stimulated as indicated. Y.p., Y. pseudotuber-culosis, MOI 5. Shown are 10-min frames from time-lapse microscopy performed at 40× magnification of annexin V and PI dual staining during the pro-gression of cell death. Twelve frames preceding membrane rupture are shown to depict the 2 h of cell-death progression leading to loss of membraneintegrity. (Scale bar: 10 μm.) (F and G, Upper) Cells stained with wheat germ agglutinin, annexin V, and PI were imaged at 4× magnification at 2-min intervals,with 4,000 cells per field of view. Annexin V and PI signals were extracted from wheat germ agglutinin-labeled cells, converted into numerical values, andplotted on the y axis for annexin V and on the x axis for PI. Representative time points of 0, 1.5, and 3 h are shown. (Lower) Cartoons depict our simplifiedmodel of the progression of phosphatidylserine externalization and membrane rupture during necroptosis or LPS/5z7-driven cell death. All imaging ex-periments are representative of three or more experiments.

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To identify caspases responsible for the cleavage of GSDMDand GSDME, we stimulated macrophages from mice with vari-ous combinations of caspase knockouts with LPS/5z7 to observegasdermin cleavage as well as cell death. We found that all threeGSDMD cleavage products were abolished in Rip3−/− Casp8−/−

macrophages (Fig. 4F). The p43 subunit is no longer present inCasp3−/− and Casp3−/−7−/− macrophages with heightened pres-ence of the p30 fragment. All three fragments are present inthe Casp9−/− macrophages. Casp1−/−11−/− and Asc−/− macrophagesshow a minor or no decrease in the appearance of p30 (Fig. 4F).The complete loss of all three fragments in the Rip3−/− Casp8−/−

macrophages in conjunction with the minor loss of the p30 fragmentin Casp1−/−11−/− and Asc−/− macrophages shows that caspase-8is necessary for the generation of active GSDMD during TAK1inhibition.In GSDME cleavage, as in GSDMD cleavage, the p30 fragment

is absent in Rip3−/− Casp8−/− macrophages. The p30 fragmentis mostly abolished in Casp3−/− macrophages and is completelyabolished in Casp3−/−7−/−macrophages (Fig. 4F). Therefore caspase-8

drives GSDME cleavage primarily via caspase-3 with minor a con-tribution from caspase-7, in agreement with published work (37).Testing the caspase-knockout macrophages for cell death, we ob-

served mild kinetic delays in Casp3−/−7−/−, Casp9−/−, Casp1−/−11−/−,and Asc−/− macrophages (Fig. 4G). These delays in cell death areconsistent with the model of overlapping functions of multipleeffector proteins in driving cell death. Caspase-8 is the criticalinitiator that drives both arms of gasdermin activation, generatingp30 fragments of GSDMD and GSDME. Casp9−/− shows no de-fect in the cleavage of either gasdermin but does display a milddelay in cell-death kinetics. Caspase-9 may drive mitochondrialdysfunction and reactive oxygen species accumulation, which hasbeen previously reported during TAK1 inhibition (9, 38).

IL-1 Secretion Requires Two Distinct Cell Populations to GenerateSignal 1 and Signal 2. NLRP3 activation via potassium efflux asa consequence of membrane pore formation has recently beenshown to occur during both pyroptosis and necroptosis (22, 36).Potassium efflux-induced inflammasome activation thus may be acommonality shared among various necrotic modes of cell death(39). We therefore wanted to know if TAK1 inhibition-inducedcell death, with its resemblance to pyroptosis in morphology, hasthe ability to induce inflammasome activation and IL-1 release.Stimulation of BMDMs simultaneously with LPS/5z7 or with

LPS alone did not lead to the release of IL-1 in the supernatant(Fig. 5A), perhaps unsurprisingly because of the requirement ofMAPK and NFκB signaling for pro–IL-1 synthesis, which wouldbe inhibited with 5z7. Much like IL-1, TNF is undetectable whenTAK1 is inhibited by 5z7 (Fig. 5B). Using Rip3−/− Casp8−/−

macrophages to bypass cell death, we found that 5z7 is still ef-fective in blocking all pro–IL-1β induced by LPS stimulation(Fig. 5C). Similarly, at a Y. pseudotuberculosis multiplicity ofinfection (MOI) that is effective in inducing RIP3/caspase-8–dependent cell death (MOI 15), we find that pro–IL-1β synthesisis sufficiently blocked in the presence of YopJ (Fig. 5D).Interestingly, and unlike agonist-driven cell death, during low-

MOI Yersinia infection IL-1β and IL-1β are found in the super-natant in a RIP3/caspase-8–dependent manner (Fig. 5E). Weobserved an inverse correlation between IL-1β abundance andMOI, with lower Yersinia MOI correlating with higher cytokinelevels in the supernatant (Fig. 5E). Of note, at the doses at whichabundant IL-1β secretion is observed, cell death as measured byPI+ nuclei approaches the lower limit of detection (Fig. 5F).Peterson et al. have shown with TNF that during Yersinia in-fection, because the bacterial population does not uniformlytranslocate adequate levels of YopJ to fully block TAK1 activity,insufficiently TAK1-inhibited cells retain the ability to synthesizecytokines (40). Similarly, we observe that cellular pro–IL-1β issynthesized when the macrophage population is challenged withdiminishing doses of YopJ-sufficient Yersinia (Fig. 5G).IL-1β maturation requires an additional inflammasome signal

to activate caspase-1. Given that IL-1β release is both inverselyrelated to cell death and requires cell death for its maturation,we hypothesized that two cellular populations are needed togenerate the two signals required for IL-1β production duringYersinia infection. We propose that pro–IL-1β is synthesized incells that still retain TAK1 function. This population could beexperiencing TLR activity via contact with less virulent bacteriain the inoculum or with bacterial-shed outer membrane vesicles(41). The inflammasome signal is provided by the TAK1-inhibited cells that are unable to synthesize cytokines but aredriven to necrotic cell death via caspase-8–induced GSDMDactivation. Upon the death of TAK1-inhibited cells, the releaseof active inflammasome results in the cleavage and maturation ofIL-1β in pro–IL-1β–sufficient cells. Our line of thinking is sup-ported by reports that oligomerized ASC and the functionalNLRP3 inflammasome can have extracellular activity (42). Al-ternatively, the dying population of macrophages may be exposing

A B C

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E

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Fig. 4. Caspase-8 induces GsdmD cleavage to drive pyroptosis, curtailingapoptosis. (A) Cleavage products of GsdmD and GsdmE in cell lysate whenBMDMs are stimulated with various cell-death triggers or Yersinia infection.The time point shown is 3 h post stimulation or infection with Y. pseudo-tuberculosis at an MOI of 25. (B) Cell-death kinetics by PI incorporation inB6 and Gsdmd-/− BMDMs stimulated with LPS/5z7. (C) Cell-death kinetics byPI incorporation in B6, Gsdmd-/−, and Rip3−/−Casp8−/− BMDMs infected withYersinia (MOI 25). (D) Time-lapse microscopy of B6 and GsdmD−/− BMDMstriple stained with Neuro-DiO, annexin V, and PI imaged at 20× magnifica-tion. Cells were stimulated with LPS/5z7. Image series depict the hourleading up to membrane rupture in GsdmD−/− BMDMs. (Scale bars: 10 μm.)(E) Time-lapse microscopy of B6 and GsdmD−/− BMDMs triple stained withannexin V and PI imaged at 40× magnification. Cells were infected withYersinia (MOI 5). Imaged series depict the 3 h leading to membrane rupturein GsdmD−/− BMDMs. (Scale bars: 10 μm.) (F) Cleavage products of GsdmDand GsdmE in cell lysate of BMDMs from various knockouts when stimulatedwith LPS/5z7 for 2 h. (G) LPS/5z7-driven cell-death kinetics by PI incorpora-tion in BMDMs deficient for various caspases or ASC. The cytotoxicity curveof each genotype is superimposed over B6 cytotoxicity curves for clarity ofcomparison. All kinetic cytotoxicity data, Western blots, and imaging experi-ments are representative of three or more experiments.

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endogenous oxidized phospholipids from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) (also known as “oxPAPC”),which has recently been shown to be a potent inducer of IL-1production in both macrophages and dendritic cells (43).We tested this model by artificially generating two populations

during LPS/5z7 treatment (Fig. 5 H–J). LPS serves as signal 1 togenerate pro–IL-1 synthesis. Six hours later, 5z7 is added to thecells alone or in combination with a second population of naivecells (Fig. 5H). We found that, unlike cells treated simulta-neously with LPS and 5z7, cultures in which cells experienced

sequential LPS treatment followed by TAK1 inhibition releasedIL-1β into the supernatant. This effect is even more pronouncedwhen the second cell population was added simultaneously with5z7 (Fig. 5I).Taking this one step further, we used Rip3−/− Casp8−/− mac-

rophages as the LPS-primed population. Six hours after LPSstimulation, B6 BMDMs were added in the presence or absenceof 5z7. Significantly elevated supernatant IL-1β was detected inmixed cultures that contained LPS-activated Rip3−/−Casp8−/−

cells and B6 cells in the presence of 5z7. Mixed cultures con-taining LPS-activated Rip3−/−Casp8−/− cells and B6 cells in theabsence of 5z7 did not produce significantly more IL-1β thanhomogeneous Rip3−/− Casp8−/− cell cultures stimulated with LPSalone (Fig. 5J). Our findings indicate that both a population ofactively pyroptotic cells and a population of non-TAK1 inhibitedcells are needed for IL-1 release.

Human Macrophages Are Resistant to TAK1 Inhibition-Induced CellDeath with No Consequent Secretion of IL-1β. In mouse macro-phage cultures, YopJ-dependent cell death is observable across arange of bacteria MOIs. At high MOI, YopJ-independent celldeath predominates, likely due to bacterial overload. YopJ-specific cell death is apparent at MOIs lower than 60, is optimalat MOIs around 30, and at lower doses approaching backgroundlevels (Fig. 6A). Human cells, in contrast, do not display a YopJ-dependent cell-death process at any MOI tested. We challengedhuman peripheral blood mononuclear cell (PBMC)-derivedmacrophages (Fig. 6B), the monocyte-like cell line U937 (SIAppendix, Fig. S5A), and monocyte-derived macrophages (SIAppendix, Fig. S5B). In all cases, bacterial outgrowth-inducedcytotoxicity is readily observed at an MOI of 120. However, wewere unable to detect YopJ-dependent cytotoxicity when cellswere challenged with wild-type Y. pseudotuberculosis or the ΔyopJmutant at MOIs ranging from 120 down to 15 (Fig. 6B and SIAppendix, Fig. S5 A and B). Similarly, human cells were resistantto killing by LPS/5z7 (Fig. 6 C and D), as is consistent withpreviously published work (38). With the additional treatmentof zVAD, human cells underwent rapid necroptosis with dualannexin V and PI positivity (Fig. 6 C and D). With respect tocytokine production, the presence of YopJ reduces pro–IL-1βsynthesis by human PBMC-derived macrophages (Fig. 6E, Left).Similarly, 5z7 effectively blocked LPS-induced pro–IL-1β synthesis(Fig. 6E, Right) and TNF production from PBMC- and monocyte-derived macrophages (Fig. 6G and SI Appendix, Fig. S5D). IL-1βlevels in the supernatants of Yersinia-infected human PBMC- andmonocyte-derived macrophages exposed to Yersinia infection areat the level of LPS-induced IL-1 secretion (Fig. 6G and SI Ap-pendix, Fig. S5C). Lower MOIs did not increase IL-1 release intothe supernatant, as is consistent with the lack of cell death-relatedinflammasome activation. In contrast, TNFα production afterchallenge with YopJ-sufficient Y. pseudotuberculosis did increasewith diminishing MOI (Fig. 6G and SI Appendix, Fig. S5D), con-sistent with the findings of Peterson et al. (40) that reduced YopJefficacy allows break-through of NFκB activity.We considered the possibility that human macrophages may

be able to up-regulate prosurvival factors more quickly with LPSstimulation, which would allow the cells to survive simultaneousLPS + 5z7 treatment. Indeed, when human PBMC-derived mac-rophages were pretreated for 2 h with 5z7 and then were stimulatedwith LPS, we were able to detect cell death (Fig. 6H and SI Ap-pendix, Fig. S5E) as well as caspase activation and GSDME cleav-age (Fig. 6I). Of note, we were unable to detect the p30 fragment ofGSDMD. However, even with 5z7 pretreatment, the percentage ofcell death was only modest, suggesting that prosurvival factors maybe able to bypass TAK1 inhibition in human cells.

A

D

F

I J

G

H

E

B C

Fig. 5. IL-1 secretion requires two distinct cell populations to generatesignal 1 and signal 2. (A) Supernatant IL-1α and IL-1β measured by ELISA 6 hpost stimulation by LPS/5z7 compared with canonical inflammasome stimuli.(B) TNF secretion measured by ELISA 6 h post stimulation by LPS/5z7 comparedwith canonical inflammasome stimuli. (C and D) Pro–IL-1β from cell lysates ofRip3−/−Casp8−/− BMDMs stimulated with agonists (C) or infected with Yersinia(MOI 15) (D) at the indicated time points. (E) Supernatant IL-1α andIL-1βmeasured by ELISA 16 h post Yersinia infection of B6 and Rip3−/−Casp8−/−

BMDMs as a function of decreasing bacterial MOI. (F) Yersinia YopJ-inducedcytotoxicity in B6 and Rip3−/−Casp8−/− BMDMs at 6 h post infection as a func-tion of decreasing bacterial MOI. (G) YopJ-sufficient Yersinia infected at de-creasing MOIs. Pro–IL-1β synthesis was measured from cell lysate of B6 andRip3−/−Casp8−/− BMDMs (16 h). (H) Experimental schematics for I and J. (I) IL-1βELISA from B6 BMDMs asynchronously stimulated with the indicated signalswith a 1-h interval between the first and second stimulus. The last two con-ditions represent the addition of B6 BMDMs and 5z7 for the second signal.(J) IL-1β ELISA from mixed cultures of B6 and Rip3−/−Casp8−/− BMDMs asyn-chronously stimulated with the indicated signals with 1 h between the firstand second stimulus. The first population of cells consists of Rip3−/−Casp8−/−

BMDMs plated overnight and experiencing the first signal for 1 h before theaddition of the second signal. The second signal is either absent or is presentin B6 BMDMs with or without 5z7. Time-point cytotoxicity quantificationsand ELISA data are shown as ± SD from three independent experimentscompared using Student’s two-tailed t test: ns, nonsignificant (P > 0.05); *P < 0.05;**P < 0.01; ***P < 0.001. All Western blot data are representative of threeor more experiments.

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DiscussionIn the present study, we demonstrate that Yersinia YopJ-inducedmurine macrophage death involved caspase-8–induced cleavageof both GSDMD and GSDME. The ensuing cell death is rapid,morphologically pyroptotic, and induces IL-1 release (Fig. 7). Atthe time of the writing this paper, work from the Kannegantilaboratory reported that the genetic ablation of TAK1 frommyeloid cells resulted in spontaneous cell death during macro-phage maturation (44). Their work shows that loss of TAK1 leadsto spontaneous cell death, NLRP3 activation, and IL-1 productionin macrophages due to tonic TNF signaling. The genetic ablation ofTAK1 by the Kanneganti laboratory complements our work with

Yersinia infection and acute TAK1 inhibition with 5z7, providingfurther evidence for the central role of TAK1 in cell death andinflammation.GSDMD is a critical effector of pyroptosis which disrupts cell

membranes upon cleavage by caspase-1/-11 (12, 36, 45). Simi-larly, GSDME is an effector driving necrosis downstream ofcaspase-3 (33, 46). These findings prompted the Shao group toredefined pyroptosis as cell death mediated by gasdermin acti-vation (47). Our work extends these studies and shows that ac-tivation of caspase-8 in the context of TAK1 inhibition results incleavage of GSDMD, leading to a pyroptotic-like cell death.Further study is needed to determine whether caspase-8 can cleaveGSDMD directly or if other intermediary substrates independent of

A B

C D

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H

I

Fig. 6. Human macrophages are resistant to TAK1 inhibition-induced cell death with no consequent secretion of IL-1. (A) Yersinia-induced murine mac-rophage cell death at various bacterial MOIs; MOIs of 60–30 show YopJ-specific cell death. (B) Yersinia-induced death of human monocyte-derived macro-phages. Bacteria were infected at multiple MOIs with no YopJ-dependent killing observed. (C) Human PBMC-derived macrophages were treated with LPS,LPS/5z7, or 5z7 in the presence or absence of zVAD (50 μM). Kinetics of cell death was measured by PI+ nuclei. (D) Representative images of triple-stainedhuman PBMC-derived macrophages taken at 4× magnification. PBMC-derived macrophages were stimulated as in C. The image shown is at 7.5 h poststimulation. (Scale bar: 100 m.) (E) Pro–IL-1β from cell lysates of human PBMC-derived macrophages challenged with Yersinia at the indicated MOI and timepoints (Left) or stimulated with LPS ± 5z7 (Right). (F and G) Supernatant IL-1β (F) and TNF (G) from human PBMC-derived macrophages stimulated with LPS/5z7 or Yersinia at MOIs that elicit robust IL-1α/β secretion from murine macrophages. (H) Human PBMC-derived macrophages were stimulated with theindicated conditions and kinetically imaged with triple staining (Neuro-DiO, PI, and annexin V). The second row from the bottom involved a 2-h5z7 pretreatment before LPS stimulation. (I) Human PBMC-derived macrophages were stimulated with the indicated conditions for 4 h and were ana-lyzed by Western blotting for caspase-8 and caspase-3 cleavage as well as GSDMD and GSDME. The second-to-last condition involved 2 h of 5z7 pretreatmentbefore LPS stimulation. All kinetic cytotoxicity data, Western blots, ELISAs, and imaging experiments are representative of three or more experiments.

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casppase-1/11 are required to generate the pore-forming p30 sub-unit. Additionally, the cell-death response is only kinetically delayedin Gsdmd−/− macrophages, suggesting that the activation of othercell-death effectors may be engaged to ensure the eventual demiseof the cell under TAK1 inhibition.Gasdermin cleavage may have functions beyond cellular oblit-

eration. Work by both the Shao and Lieberman laboratories ele-gantly demonstrated that cleaved GSDMD can attack bacteriaand reduce their viability (48). Other work by the Miao laboratoryhas shown that pyroptosis can result in pore-induced intracellulartraps, leading to the retention of intracellular bacteria within thecarcass of dying cells to facilitate phagocytic clearance (49). Jor-gensen’s work places the importance of macrophage pyroptosisinto an in vivo context, in which IL-1 production during pyroptosisrecruits secondary phagocytic cells to the site of infection to engulfdead cell carcasses along with its damaged bacterial load.During traditionally pyroptotic infections such as with Legion-

ella, Salmonella, Francisella, and Burkholdia, IL-1 maturation isintrinsic to the infected, dying cell (50). The case of Yersinia in-fection is particularly interesting, since MAPK signaling is inhibitedby YopJ, effectively blocking pro–IL-1 synthesis in doomed, infectedmacrophages (30, 31). Since bacterial populations are not homog-enous in their virulence, some percentage of macrophages at thesite of infection will not be sufficiently intoxicated by YopJ, thusretaining the ability to synthesize MAPK-dependent cytokines. Wepostulate that these MAPK-competent cells are also the producersof pro–IL-1, with neighboring pyroptosing cells serving as platformsfor potassium efflux-induced inflammasome formation (Fig. 7). Wepredict that the MAPK-competent cells then complete the process-ing of IL-1 maturation by efferocytosing dead cells or by acquiringextracellular ASC/NLRP3 inflammasomes released by dying cells.Alternatively, the live macrophages may be sensing endogenousoxidized lipids, such as oxPAPC from dying cells, which are knownto induce IL-1 production in macrophages and dendritic cells viaactivation of caspase-11 (51).

Taken together, these findings indicate that macrophages thatare defective for the cell-death response, such as Rip3−/−

Casp8−/− and RIP1 kinase dead (RIP1K45A), would therefore bedefective in IL-1 maturation as well, making the animals moresusceptible to uncontrolled Yersinia replication (15). Extendingthis line of argument, we found that human macrophages withminimal production of IL-1 during Yersinia infection do notundergo YopJ-dependent cell death, an observation that mayprovide some insight into the devastation caused by the plague.In the present study, we used IP2666 Y. pseudotuberculosis, whichexpresses hexaacylated LPS capable of triggering TLR4. Themore dangerous Y. pestis evolved from Y. pseudotuberculosis andin the process acquired a hypoacylated TLR4-evasive LPS (52).Additionally, hypoacylated lipid IVa acts as an antagonist forTLR4 activation in human macrophages, although it is weaklystimulatory against TLR4 of murine macrophages (53). With YopJinhibition of TAK1 tolerated by human macrophages, in combi-nation with a TLR-4–evasive LPS structure, Y. pestis effectivelycreates a silent infection. This point has been elegantly demon-strated by the forced expression of Escherichia coli LPS on theKim4 strain of Y. pestis as an attenuation strategy (54).Other examples of necrotic forms of cell death lending pro-

tection to the infected host can be found in studies of nec-roptosis. For instance, RIP3 has been shown to be protective inthe case of vaccinia virus infection and CMV infection (55) andduring HSV replication by the induction of cell death in infectedmouse cells (56). In the case of HSV, necroptosis in mouse cellslimits viral replication, while the absence of necroptosis in hu-man cells leads to uncontrolled viral replication. These findingswith HSV bear striking similarity to the differential species-basedcell-death responses and pathogen replication highlighted inour study.Human macrophages therefore may have an inborn tolerance

to highly pathogenic bacterial neighbors, to the detriment of theorganism. At the macrophage level, the resistance to cell deathmay lie in the higher expression or longer half-life of key pro-survival proteins. In mouse macrophages, complex IIa formswhen there are inadequate prosurvival factors, including cellularFLICE-inhibitory protein (cFLIP) (57) and inhibitors of apo-ptosis (IAPs) (25). More recent work has also implicated the lossof IAP and cFLIP during TAK1 inhibition as a reason for celldeath (58). IAPs and FLIP are top candidates in the in-vestigation of how the regulation of these proteins results in in acell-death defect in human macrophages.Very recent investigations in the field of cell death also show

that MAPK-activated protein kinase 2, a downstream target ofP38 and TAK1, is required for RIP1 phosphorylation at Ser321(59). This MAPK-driven RIP1 phosphorylation inhibits complexII formation and thus limits cell death. The state of RIP1 regu-lation in human macrophages may also be an area of interest.The differential response of human and murine macrophages

demonstrated by our study and others (56) thus calls attention tothe need to study both mouse and human cells to understand thecourse of illness in humans, especially when rodents are reser-voirs for the human disease of interest. Beyond Yersinia, theanthrax toxin and lethal factor effectors of Bacillus anthracisinhibit MAPK activation and have been observed to elicit celldeath and IL-1 production (60). Investigating the role of gas-dermins and the presence of pyroptotic features in this and otherinfectious systems known to inhibit MAPK or NFκB signalingmay provide a deeper understanding of the mechanism andscope of necrotic cell death in host defense.

Materials and MethodsWild-type and ΔyopJ IP2666 Y. pseudotuberculosis strains were gifts fromRalph Isberg, Tufts University School of Medicine, Boston, and Joan Mecsas,Tufts University School of Medicine, Boston, and were grown on LB platescontaining Irgasan (Sigma-Aldrich). Overnight cultures grown at 26 °C were

Pro-survival genes:cFLIP, IAPsCytokines:Il1, TNF

TLR4

NF-κBMAPKs

RIP1

RIP1FADDCasp8

GsdmD

GsdmE

(Casp1/11)

Casp3/7

K+

TAK1 Inhibited cell

TAK1 Activecell

TAK1

pro-IL-1

IL-1 release

In ammasome

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ApoptoticDeathEffectors

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Fig. 7. Model of Yersinia-induced cell death and IL-1 production. Twomouse macrophages are illustrated. The macrophage on the left (a TAK1-inhibited dying cell) is sufficiently intoxicated with Yersinia YopJ to inhibitTAK1. The bacterium is also recognized by TLR4, which signals via RIP1 toform a cell-death complex composed of RIP1, caspase-8, and, likely, FADD.This death complex drives GsdmD cleavage and activation partially via cas-pase-1/11 and GsdmE cleavage via caspase-3 and caspase-7, both of whichresult in cell membrane permeabilization and subsequent cell death. Thereare likely additional effectors of cell death-driven activation, which in con-junction with GsdmD may lead to potassium flux out of the cell and sub-sequent inflammasome activation. A second macrophage that is not sufficientlyintoxicated by YopJ is necessary for the production of pro–IL-1 cytokines. Uponcontact with the dying cell or cellular components, IL-1 maturation occurs. SeeDiscussion for details.

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diluted to an OD600 of 0.2 and were grown at 26 °C for 2 h followed by 37 °Cfor 2 h. Macrophage cultures were infected at the MOI ranges indicated inthe figures and their legends. Macrophages were harvested from micehoused in a pathogen-free facility at the Tufts University School of Medicine,and experiments were performed in accordance with regulations and ap-proval of the Tufts University Institutional Animal Care and Use Committee.Statistical analysis was performed using the Student’s t test (two-tailed) us-ing GraphPad Prism; *P < 0.05; **P < 0.01; ***P < 0.001. Detailed methodsare provided in SI Appendix, SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Ralph Isberg and Dr. Joan Mecsas forthe gift of Y. pseudotuberculosis. We thank Dr. Kate Fitzgerald and Dr. EgilLien for insightful discussions and sharing of bones from various geneticknockout animals, and Dr. Yinan Gong and Dr. Brigitte Huber for sugges-tions and guidance at various phases of this project. This work was sup-ported by National Institute of Allergy and Infectious Disease (NIAID)Grant AI135369 and Russian Science Fund Project 15-15-00100 (to A.P.) andNIAID Grant T32-AI-007077 to the Immunology Graduate Program (TuftsUniversity).

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